Under normal physiological condition, angiogenesis is a process tightly regulated by several factors and it plays a vital part in repairing and maintaining the normal function of the body. However, rapid growth of blood vessels inside tumor is a common clinical phenomenon. It is demonstrated in many animal models and clinical studies that the inhibition of angiogenesis in tumor can inhibit the growth of tumor and induce the death of tumor cell, so that therapeutic effect is achieved. Therefore, the inhibition of angiogenesis has become an important trend for the development of anti-tumor drugs. Macromolecule anti-angiogenesis drugs, such as Avastin, have been approved by the FDA. There are also drugs under pre-clinical and clinical studies. In addition, new anti-angiogenesis drugs are also widely used in the treatment of angiogenesis-related diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and result in significant therapeutic effect.
Angiogenesis is a complex process regulated by many active biological factors. A key process in angiogenesis is the binding and the activation of endothelial cell surface receptor by various growth factors, which control the activity of endothelial cell via intracellular tyrosine phosphorylation signalling and improve angiogenesis. Among these growth factors, vascular endothelial cell growth factor (VEGF) is the most important factor involved in the control of angiogenesis. VEGF is the most potent and the most specific factor in the induction and improvement of angiogenesis. It is overexpressed in almost all human tumors and thus has become an important molecular target in the development of anti-tumor therapy. VEGF has many receptors on the surface of vascular endothelial cell, including VEGFR-1 (also named as Flt-1) and VEGFR-2 (also named as KDR or Flk-1). VEGFR-1 and VEGFR-2 both comprise an extracelluar portion, composed of 7 immunoglobulin-like domains (D1-D7) capable of binding VEGF, and an intracellular portion comprising tyrosine kinase group. Once the receptor is activated by VEGF, the intracellular tyrosine kinase gene is phosphorylated, which results in a signaling cascade and eventually leads to angiogenesis. Due to the importance of VEGF signaling to angiogenesis, blocking VEGF or VEGF receptor to inhibit angiogenesis has significant anticancer effect and is also important for the treatment of other angiogenesis-related diseases such as retinal vasculopathy.
The stability of fusion protein agents has an important influence on their biotechnical applications, and is also vital for the improvement of drug quality and realization of industrial production. The spacial structure of a protein changes under certain physical and chemical conditions, which in turn leads to the change in physical and chemical properties and loss of biological activity. This is called protein denaturation, which is used to describe the process that a protein changes its intramolecular structure and properties when subjected to the influence of physical or chemical factors. Detection of thermal stability of a protein can be used to determine the denaturation effectively and thus is used as a stability test for protein during the development of therapeutic proteins. The detection of thermal stability is mainly carried out by irradiating a protein sample with UV light so that the protein sample emits fluorescence, and determining the denaturation temperature (Tm) of the protein sample by measuring the wavelength and the light intensity of emitted fluorescence, which reflect the change in its structure. Also, protein aggregation is detected by UV excitation using static light scattering. The light intensity of a protein sample changes significantly between aggregation and non-aggregation states. The aggregation onset temperature (Tagg) of a protein sample can be detected accurately according to the change in light intensity. The stability of a protein is reflected by denaturation temperature (Tm) and aggregation onset temperature (Tagg) of the protein.
The constraint conditions for development of macromolecular drugs include the therapeutic effect, the stability, and the feasibility of industrial production of the therapeutic agent. It is necessary to find a drug inhibiting angiogenesis or vascular growth with determined therapeutic effect and high stability.